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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

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Research Article ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF BROMFENAC SODIUM AND MOXIFLOXACIN IN THEIR COMBINED DOSAGE FORM Pradhan Prasanna Kumar*, Solanki Kuldipsinh K, Upadhyay Umesh M Department of Quality Assurance, Sigma Institute of Pharmacy, Bakrol, Vadodara, Gujarat, India *Corresponding Author Email: prasanna.k.pradhan@gmail.com Article Received on: 10/07/14 Revised on: 03/08/14 Approved for publication: 11/08/14 DOI: 10.7897/2230-8407.0509137 ABSTRACT A simple, rapid, accurate and precise method was developed for Bromfenac sodium and Moxifloxacin. BDS Hypersil C-18 column (250 mm × 4.6 mm id) 5 μm particle size was used as stationary phase. The mobile phase used was Buffer KH2PO4: Acetonitrile : Triethylamine at pH 4.0 adjusted with Ortho phosphoric acid. The mobile phase was delivered at flow rate 1.0 ml/min. UV detection was set at 275 nm. The retention time for Bromfenac sodium and Moxifloxacin was found 4.637 minutes and 7.630 minutes respectively. The linearity was observed over the range of 2.25-6.75 mcg/ml and 12.5-37.5 mcg/ml for Bromfenac sodium and Moxifloxacin respectively. The LOD was found 0.46 mcg/ml and 0.64 mcg/ml for Bromfenac sodium and Moxifloxacin respectively; whereas LOQ was found to be 1.41 mcg/ml for Bromfenac sodium and 1.94 mcg/ml for Moxifloxacin. Moreover, the % RSD for repeatability, Inter and intra-day precision was found to be less than 2 % which reveals method is precise. The correlation co-efficient found to be 0.997 and 0.999 for Bromfenac sodium and Moxifloxacin respectively. The % recovery was found to be 99.79-100.09 % for Bromfenac sodium and 99.90-100.03 % for Moxifloxacin. The assay percentage was found to be 98.65 % and 97.50 % for Bromfenac and Moxifloxacin respectively. All the validation parameters were checked according to ICH guidelines finally it is concluded that the developed method is precise and accurate. Keywords: Bromfenac sodium, Moxifloxacin, Method validation, HPLC. INTRODUCTION The aim of present work is to develop simple, rapid, precise and economical method and to validate the method according to ICH guidelies. Various articles from different journals show at the time I have started my work that there was no RP-HPLC method developed. Bromfenac sodium is non-steroidal anti inflammatory drug (NSAID) for ophthalmic use. Chemically it is known as 2-[2-amino-3-(4-bromobenzoyl)phenyl]acetic acid sodium salt. Ophthalmic NSAIDs are becoming a counterstone for management of ocular pain and inflammation. Their well-characterized anti-inflammatory activity, analgesic property and established safety record have also made NSAIDs an important tool to optimize surgical outcomes. The high degree of penetration and potency of bromfenac sodium can be attributed to halogenations of molecule: by adding bromine moiety the NSAIDs becomes highly lipophilic which allows for rapid, sustained drug levels in the ocular tissues. Moxifloxacin is an antibiotic in a group of drugs called fluoroquinolones (flor-o-KWIN-o-lones). Moxifloxacin fights bacteria in the body. Moxifloxacin is a fourth-generation synthetic fluoroquinolone antibacterial agent. It is marketed worldwide (as the hydrochloride) under the brand names Avelox, Avalox and Avelon for oral treatment. Moxifloxacin is used to treat different types of bacterial infections, Respiratory tract infections, Cellulitis, Intra-abdominal infections, Endocarditis, Meningitis and Tuberculosis. Moxifloxacin may also be used for purposes not listed in this medication guide. Chemically it is known as 1-cyclopropyl-7-[(1s,6s)-2,8-diaz[4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo-quinoline carboxylic acid1,2,4,5. MATERIALS AND EQUIPMENTS API samples were gifted by sun pharma ltd. The combination pharmaceutical dosage form used for experiment was manufactured by opticarma pharma ltd, HPLC grade

methanol (Samir Tech-Chem Pvt Ltd.), Orthophosphoric acid, Phosphate buffer, Sonicator (EIE instruments Pvt ltd.), pH meter (Welltronix PM100), Micro analytical balance (Shimadzu BL-220H), HPLC instrument (Shimadzu LC 20AT) etc were used. Optimized Chromatographic Condition The chromatographic column BDS hypersil C-18 (250 mm × 4.6 mm id × 5 μm particle size) was used as stationary phase and Buffer (0.1 M Potassium Dihydrogen Phosphate with Orthophosphoric acid, pH 4.0), Acetonitrile and Triethylamine in ratio of 55:45:0.1 %v/v/v was used. The flow rate was set at 1.0 ml/min. The column temperature maintained at 25°C. The elluent was detected at 275 nm with 20 μl of injection volume. Selection of analytical wavelength 9 mcg/ml and 50 mcg/ml solutions of Bromfenac sodium and Moxifloxacin were being made. The solutions were scanned in range of 400-200 nm. The overlay graph shows that both the drugs can significantly detected at 275 nm. Preparation of standard stock solution of Bromfenac sodium Weigh accurately 4.5 mg of Bromfenac sodium into 100 ml volumetric flask and dissolve in 10 ml of diluents. Then the flask was sonicated for 10 minutes. Volume was made up to the mark to get 45 mcg/ml solutions. From this take 1 ml to the 10 ml volumetric flask and volume was adjusted with diluents to get the final concentration of 4.5 mcg/ml. Preparation standard stock solution of Moxifloxacin Weigh accurately 25 mg of Moxifloxacin into 100 ml volumetric flask and dissolve in 10 ml of diluents. Then the flask was sonicated for 10 minutes. Volume was made up to the mark to get 250 mcg/ml solutions. From this take 1 ml to

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the 10 ml volumetric flask and volume was adjusted with diluents to get the final concentration of 25 mcg/ml. Preparation of mobile phase A mixture of 55 ml phosphate buffer, 45 ml acetonitrile and 0.1 ml of triethylamine was filtered through 0.45 μm filter paper then sonicated for 10 minutes. It was then used as mobie phase. Preparation of 0.1 M Phosphate Buffer Potassium dihydrogen phosphate 1360 mg was accurately weighed and dissolved in 100 ml of HPLC grade water pH was adjusted to 4.0 with the help of orthophosphoric acid.

Assay Prepare standard stock solutions of both the drugs as given above. Prepare sample solutions from marketed formulation; from the formulation take 0.5 ml in volumetric flask make up volume up to 10 ml from this take 1 ml make up to 10 ml which gives solution of 4.5 mcg/ml of bromfenac sodium and 25 mcg/ml of moxifloxacin. Both were measured at 275 nm and % assay is calculated. Method Validation The developed method is validated with respect to validation parameters like accuracy, precision, linearity and range, LOD and LOQ, Robustness, ruggedness, system suitability etc.

Table 1: Linearity Data of Bromfenac Sodium and Moxifloxacin

BROM MOXI Conc.(mcg/ml) Area Conc.(mcg/ml) Area 2.25 950.515 12.5 2187.756 3.375 1308.052 18.75 3142.431 4.5 1852.875 25 4272.08 5.625 2288.957 31.25 5277.285 6.75 2731.848 37.5 6292.076

SLOPE 315.9 218.5 SD 44.53 42.50

LOD 0.46 0.64 LOQ 1.40 1.94

Table 2: Intraday and Interday Precision of Brom

Concentration (μg/ml) Intraday (Area* ± SD) % RSD Interday (Area* ± SD) % RSD

2.25 929.37 ± 1.85 0.199 930.3 ± 1.84 0.198 4.5 1879.20 ± 3.74 0.199 1881.08 ± 3.73 0.198 6.75 2811.79 ± 5.598 0.199 2814.59 ± 5.61 0.199

Table 3: Intraday and Interday Precision of Moxi

Concentration (μg/ml) Intraday (Area* ± SD) % RSD Interday (Area* ± SD) % RSD

12.5 2130.32±11.51 0.54 2137.66±3.07 0.143 25 4319.54±3.899 0.09 4330.81±5.43 0.125

37.5 6467.69±7.296 0.11 6475.03±6.74 0.104

Table 4: Repeatability Data of Bromfenac Sodium and Moxifloxacin

BROM MOXI Conc.(mcg/ml) Area Conc.(mcg/ml) Area 4.5 1884.886 25 4345.734 4.5 1888.685 25 4354.458 4.5 1892.401 25 4357.211 4.5 1886.754 25 4350.086 4.5 1890.504 25 4358.816 4.5 1894.331 25 4354.511

AVERAGE 1889.593 4353.469 SD 3.527 4.815

% RSD 0.186 0.110

Table 5: Robustness Data of Brom and Moxi

Conditioned varied Changed condition Area %RSD BROM

(4.5 μg/ml) ± SD MOXI

(25 μg/ml) ± SD BROM

MOXI

Change in flow rate of mobile phase (ml/min)

Change in flow rate 0.8 ml/min 1962.063 ± 11.43 4517.89 ± 16.91 0.582 0.374 1.2 ml/min 1848.867 ± 11.33 4258.18 ± 19.78 0.613 0.464

Change in contents of mobile phase (Buffer: Acetonitrile :Triethylamine55:45:0.1%v/v/v) Change in mobile phase 57:47 1847.01 ± 11.33 4253.05 ± 18.68 0.613 0.439

53:43 1939.24 ± 11.39 4465.86 ± 17.58 0.587 0.393 Change in pH of the mobile phase

Change in pH mobile phase 4.2 1809.61 ± 11.275 4164.57 ± 17.17 0.623 0.412 3.8 1940.54 ± 12.38 4471.89 ± 23.87 0.638 0.533

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Table 6: Recovery Data of Bromfenac Sodium and Moxifloxacin

Conc.

Level (%) Amount of sample

present (µg) Amount of std added

(µg) Amount found

(µg) % Recovery mean

± % RSD BROM 80 2.25 1.8 1.80 100.09 ± 1.12

100 2.25 2.25 2.23 99.79 ± 0.65 120 2.25 2.7 2.69 99.79 ± 0.49

MOXI 80 12.5 10 9.99 100.02 ± 1.1 100 12.5 12.5 12.49 100.01 ± 0.71 120 12.5 15 14.98 99.91 ± 0.61

Table 7: System Suitability Data of Brom and Moxi

Drugs Retention time (Rt)

(minute) Tailing Factor Theoretical plates Resolution

Bromfenac Sodium (4.5 μg/ml) 4.637 1.448 7423 _ Moxifloxacin (25 μg/ml) 7.630 1.451 6277 9.970

Figure 1: Structure of bromfenac sodium4

Figure 2: Structure of moxifloxacin5

Figure 3: Overlain Spectra of Both Drugs

Figure 4: Mix Standard Chromatogram of Moxi and Brom

Figure 5: Chromatogram of Test Samples

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Figure 6: Linearity Chart of Bromfenac Sodium

Figure 7: Linearity Chart of Moxifloxacin

Figure 8: Overlain Chromatogram of Linearity of Both Drugs

RESULTS Initially solubility test were conducted in various solvents and finally mobile phase was selected from it. To optimized chromatographic condition several trials were taken for system suitability. The graphs given below give idea about it. The assay was performed under optimized condition which gives chromatographs. The mobile phase used was Buffer KH2PO4: Acetonitrile: Triethylamine at pH 4.0 adjusted with Ortho phosphoric acid. The mobile phase was delivered at flow rate 1.0 ml/min. UV detection was set at 275 nm. The retention time for Bromfenac sodium and Moxifloxacin was found 4.637 minutes and 7.630 minutes respectively. The linearity was observed over the range of 2.25-6.75 mcg/ml and 12.5-37.5 mcg/ml for Bromfenac sodium and Moxifloxacin respectively. The LOD was found 0.46 mcg/ml and 0.64 mcg/ml for Bromfenac sodium and Moxifloxacin respectively; whereas LOQ was found to be 1.41 mcg/ml for Bromfenac sodium and 1.94 mcg/ml for Moxifloxacin. Moreover, the % RSD for repeatability, Inter and intra-day precision was found to be less than 2 % which reveals method is precise. The correlation co-efficient found to be 0.997 and 0.999 for Bromfenac sodium and Moxifloxacin respectively. The % recovery was found to be 99.79-100.09

% for Bromfenac sodium and 99.90-100.03 % for Moxifloxacin. The assay percentage was found to be 98.65 % and 97.50 % for Bromfenac and Moxifloxacin respectively. All the validation parameters were checked according to ICH guidelines. The tables contain data of Assay, Precision, repeatability, Robustness, % Recovery and system suitability. DISCUSSION The assay was performed on the eye drops and % drug contents found to be 98.65 % and 97.50 % for Bromfenac sodium and Moxifloxacin respectively. Limit for precision is % RSD should not be more than 2 %. Limit for repeatability % RSD should not be more than 2 %. Limit for robustness % RSD should not more than 2 %. Limit for % Recovery % RSD should not more than 2 %. N = 3 determinations of 3 concentrations. CONCLUSION It is concluded that the developed method is simple, rapid, accurate and precise. All the validation parameters checked according to ICH guidelines and they were in the limits so it is concluded that the developed method is simple, precise and rapid. It is also concluded that this method can be used to

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analyze the drugs in bulk as well as in combination and also can be used in industry. ACKNOWLEDGEMENT Behind every success there are lot many efforts, but efforts are fruitful due to hands making the passage smoother. I express my deep sense of gratitude for hands, people extended to me during my work. A successful outcome in any research endeavor attributes itself to the selfless guidance of the mentor. REFERENCES 1. Ahuja S and Rasmussen HT. HPLC Method Development for

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Cite this article as: Pradhan Prasanna Kumar, Solanki Kuldipsinh K, Upadhyay Umesh M. Analytical method development and validation for simultaneous estimation of bromfenac sodium and moxifloxacin in their combined dosage form. Int. Res. J. Pharm. 2014; 5(9):671-675 http://dx.doi.org/10.7897/2230-8407. 0509137

Source of support: Nil, Conflict of interest: None Declared